Download Taxonomic probes

Monitorización de
GEMs en el
ambiente.
Marcadores
Why monitor domesticated
microbial inoculants in nature?
• Risk assessment of GMMs
• Performance/behaviour studiesAg/Biotech. applications
– Biological pesticides
– Bioremediation
– Biological fertilizers (Rhizobia)
• Basic studies of microbial ecology
Questions to address:
• How many cells are present?
• Are the cells alive?
• Are the cells metabolically active?
• How are the cells distributed?
• Can the cells perform their intended tasks?
• What effect do the cells have on the natural
microbial diversity?
Marker Genes
Molecular Probes
Marker genes as specific monitoring tools- I
- LacZ protein (b-Galactosidase)
Well studied and widely used
Activity absent in Pseudomonadaceae
Different substrates: X-Gal, ONPG, MUG
Background activity
Visible only in big amounts of cells (colonies)
- XylE protein (Catecol 2-3 dioxigenase)
Detection of life cells
Impredictability (inactivated by O2…)
- LacZ protein
- XylE protein
Marker genes as specific monitoring tools- II
- Firefly luciferase (luc) or bacterial luciferase (luxAB)
Monitor metabolically active cells in the population
Detect light emission
- Luminometry
- Microscopy + sensitive cameras
- GFP (gfp): Enumerate total cell population
Regardless of physiological status
Detect by fluorescence-based methods
- Flow cytometry
- Fluorescence microscopy
Bioluminiscencia
1. Origen eucariótico (genes luc luciérnaga)
LH2 + ATP + O2
Mg2+
luciferasa
CO2 + oxiluciferina + AMP+ luz
2. Origen bacteriano (genes lux Vibrio / Photobacterium)
FMNH2 + RCHO + O2
Mg2+
luciferasa
H2O + ROOH + FMN + luz
Bioluminiscence
luxCDABE AB code for the luciferase
CDE code for luciferin biosynthesis
Strategies:
Introduce the whole operon
Constitutively luminescent bacteria
~8kb operon, interference with FA biosynthesis
Introduce the luciferase
Luciferin has to be externally added
Reaction always depends on reducing power ->
cell status
Fluorescencia
Green fluorescent protein (GFP de Aequorea victoria)
Fluorescencia verde al excitarse con luz UV o
azul- sin sustrato ni cofactor
gfp/luxAB-tagged
bacteria
Confocal microscopy
Cryosection
Nycodenz density
gradient
Fluorescence stereomicoscopy
Bacterial fraction
Flow cytometry
(gfp-tagged cells)
Luminometry
(lux-tagged cells)
9
8
8
7
7
6
6
5
5
-1
9
Log quanta sec g soil (dry weight)
Log fluorescent cell number
-1
g soil (dry weight)
P. fluorescens SBW25 in soil
4
8
12
16
20
Time (Days)
24
28
32
-1
0
Confrontation studies with antagonistic
fungal strains Trichoderma harzianum - GFP
Marker Genes: monitorisation of E.
Coli-GFP colonisation in whole animals
E.coli-GFP infecting peritoneal cavity
Marker Genes
Molecular Probes
Molecular probes to detect GEMs
Immunological techniques
DNA probes
PCR-based methods
Immunological techniques
- Fluorescent microscopy (single cells)
- ELISA (>100 cells)
Advantages:
Highest specificity (serotyping)
Detection at single-cell stage
Drawbacks:
Cross-reaction
Auto-fluorescence
Epitope expression
Rhizobium sp.
Bradirhizobium sp.
DNA probes
- Taxonomic probes
- Phylogenetic probes
Advantages:
Taxonomic level specificity
Sensitivity of 16S probes
Direct detection of interesting activities
Drawbacks:
Specificity > species level
Crossreaction (diversity unknown)
16S RNA
Fluorescence in situ hybridization (FISH)
FISH
Taxonomic
probes
In situ hybridization of a
vertical biofilm slice with a
NIT3-labeled probe specific
for the genus Nitrobacter (red
stain cluster) correlated to
oxygen and nitrate gradients
measured by
microelectrodes.
FISH
20 µm
Functional
probes
Confocal microscopic
image of a bacterial
aggregate thin
section after
hybridization with a
Cy3-labeled probe
specific for nitriteoxidizing Nitrospira
sp. (red) and a Cy5labeled probe
specific for ammoniaoxidizing Nitrosospira
sp. (blue).
PCR-based methods
- PCR --> RFLP
Advantages:
Highest sensitivity (1 cell/gr.)
In situ detection of activity
Drawbacks:
Inspecificity
Contamination
Interference of humic substances
Alterations due to sample purification
T-RFLP
(Terminal-Restriction Fragment Length Polymorphism)
Total soil DNA
PCR 16S rRNA genes
•Eubacterial primers
•5´primer fluorescent
Restriction digestion
Separation on sequencing gel